Introduction Resistance to antibiotics is a threat confronting health care system worldwide. Nurses play a significant role in combating this threat. Objectives The present study examined the knowledge and attitude of nurses towards antibiotic use and prevention of antibiotic resistance. Methods The research involved a cross-sectional study conducted in a multi-cultural tertiary healthcare setting. The participants were 341 nurses. A structured self-administered questionnaire with a good validity and reliability (α = 0.7) was used. Results The nurses surveyed showed moderate awareness of antibiotic resistance and a fair attitude towards its prevention. There was no significant correlation of demographic features with their overall knowledge and attitude ( p > 0.05). Conclusions Nurses perform a crucial part in infection control, but often lack knowledge of methods to prevent antibiotic resistance. This paucity highlights the importance of tailored interventions to help nurses improve their awareness of antibiotic resistance and create a favorable attitude towards its prevention.